scholarly journals Accumulation of Temozolomide-Induced Apoptosis, Senescence and DNA Damage by Metronomic Dose Schedule: A Proof-of-Principle Study with Glioblastoma Cells

Cancers ◽  
2021 ◽  
Vol 13 (24) ◽  
pp. 6287
Author(s):  
Lea Beltzig ◽  
Björn Stratenwerth ◽  
Bernd Kaina
Keyword(s):  
Cumulative Dose ◽  
Low Dose ◽  
Dose Range ◽  
Dose Schedule ◽  
Dna Double Strand Breaks ◽  
Glioblastoma Cells ◽  
Response Curves ◽  
Therapy Targets ◽  
Induced Apoptosis

Temozolomide (TMZ), a first-line drug in glioma therapy, targets the tumor DNA at various sites. One of the DNA alkylation products is O6-methylguanine (O6MeG), which is, in the low dose range of TMZ, responsible for nearly all genotoxic and cytotoxic effects relevant for cancer therapy. There is, however, a dispute regarding whether the TMZ concentration in the tumor tissue in patients is sufficient to elicit a significant cytotoxic or cytostatic response. Although treatment with TMZ occurs repeatedly with daily doses (metronomic dose schedule) and in view of the short half-life of the drug it is unclear whether doses are accumulating. Here, we addressed the question whether repeated low doses elicit similar effects in glioblastoma cells than a high cumulative dose. We show that repeated treatments with a low dose of TMZ (5 × 5 µM) caused an accumulation of cytotoxicity through apoptosis, cytostasis through cellular senescence, and DNA double-strand breaks, which was similar to the responses induced by a single cumulative dose of 25 µM TMZ. This finding, together with the previously reported linear dose–response curves, support the notion that TMZ is able to trigger a significant cytotoxic and cytostatic effect in vivo if the low-dose metronomic schedule is applied.

scholarly journals Cytotoxic and Senolytic Effects of Methadone in Combination with Temozolomide in Glioblastoma Cells

2020 ◽  
Vol 21 (19) ◽  
pp. 7006
Author(s):  
Bernd Kaina ◽  
Lea Beltzig ◽  
Andrea Piee-Staffa ◽  
Bodo Haas
Keyword(s):  
Pain Treatment ◽  
Human Fibroblasts ◽  
Methadone Treatment ◽  
Fibroblast Cell ◽  
Glioblastoma Cells ◽  
First Line ◽  
Induced Apoptosis ◽  
Methadone Concentration

Methadone is an analgesic drug used for pain treatment and heroin substitution. Recently, methadone has been proposed to be useful also for cancer therapy, including glioblastoma multiforme (GBM), the most severe form of brain cancer, because experiments on cultured glioma cells treated with doxorubicin showed promising results. Doxorubicin, however, is not used first-line in GBM therapy. Therefore, we analyzed the cytotoxic effect of methadone alone and in combination with temozolomide, a DNA-alkylating drug that is first-line used in GBM treatment, utilizing GBM-derived cell lines and a human fibroblast cell line. We show that methadone is cytotoxic on its own, inducing apoptosis and necrosis, which was observed at a concentration above 20 µg/mL. Methadone was similar toxic in isogenic MGMT expressing and non-expressing cells, and in LN229 glioblastoma and VH10T human fibroblasts. The apoptosis-inducing activity of methadone is not bound on the opioid receptor (OR), since naloxone, a competitive inhibitor of OR, did not attenuate methadone-induced apoptosis/necrosis. Administrating methadone and temozolomide together, temozolomide had no impact on methadone-induced apoptosis (which occurred 3 days after treatment), while temozolomide-induced apoptosis (which occurred 5 days after treatment) was unaffected at low (non-toxic) methadone concentration (5 µg/mL), and at high (toxic) methadone concentration (20 µg/mL) the cytotoxic effects of methadone and temozolomide were additive. Methadone is not genotoxic, as revealed by comet and γH2AX assay, and did not ameliorate the genotoxic effect of temozolomide. Further, methadone did not induce cellular senescence and had no effect on temozolomide-induced senescence. Although methadone was toxic on senescent cells, it cannot be considered a senolytic drug since cytotoxicity was not specific for senescent cells. Finally, we show that methadone had no impact on the MGMT promoter methylation. Overall, the data show that methadone on glioblastoma cells in vitro is cytotoxic and induces apoptosis/necrosis at doses that are above the level that can be achieved in vivo. It is not genotoxic, and does not ameliorate the cell killing or the senescence-inducing effect of temozolomide (no synergistic effect), indicating it has no impact on temozolomide-induced signaling pathways. The data do not support the notion that concomitant methadone treatment supports temozolomide-based chemotherapy.

scholarly journals Anti-apoptotic effects of 3,5,3′-tri-iodothyronine in mouse hepatocytes

2006 ◽  
Vol 191 (2) ◽  
pp. 447-458 ◽  
Author(s):  
O A Sukocheva ◽  
D O Carpenter
Keyword(s):  
Early Stage ◽  
Dose Range ◽  
Annexin V ◽  
Intracellular Camp ◽  
Camp Content ◽  
Mouse Hepatocytes ◽  
Induced Apoptosis ◽  
Apoptotic Effects ◽  
Intracellular Alkalinization

The present study demonstrates that 3,5,3′-tri-iodothyronine (T3) in physiological dose range inhibits tumor necrosis factor α(TNFα)/Fas-induced apoptosis in mouse hepatocytes. T3 pretreatment prevented Fas-induced early stage of apoptosis signs assessed by flow cytometry analysis of the annexin V positive cell population. T3 attenuated TNFα/Fas-induced cleavage of caspase-8 and DNA fragmentation. We found that T3 exerted its anti-apoptotic effects by mobilization of several non-genomic mechanisms independent of transcriptional activity. Inhibition of protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and Na+/H+ exchanger blocked T3-dependent anti-apoptotic effects indicating an involvement of these intracellular targets into T3-induced signaling cascade. Furthermore, physiological concentrations of T3, but not reverse T3, caused increases in intracellular cAMP content and activated PKA. T3 markedly induced phosphorylation of ERK. We also detected T3-dependent intracellular alkalinization that abolished TNFα-induced acidification. PKA inhibitor KT-5720 blocked T3-induced activation of ERK and intracellular alkalinization confirming the upstream position of PKA signaling. We further detected that hepatocytes from hypothyroid mice are more sensitive to TNFα/Fas-induced apoptosis than euthyroid animals in vivo. Together, these findings imply that T3 triggers PKA- and ERK-regulated intracellular pathways capable of driving and ensuring hepatocytes survival in the presence of death receptor ligand-induced damage under chronic inflammatory conditions.

Glucocorticoid Therapy Targets Proliferation, Differentiation and Bcl-2 Dependent Survival Signaling in ALL Blasts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1841-1841
Author(s):  
Peter Rhein ◽  
Stefanie Scheid ◽  
Richard Ratei ◽  
Christian Hagemeier ◽  
Karl Seeger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Induction Therapy ◽  
Molecular Mechanisms ◽  
Leukemia Cell ◽  
Apoptotic Pathway ◽  
Therapy Targets ◽  
Cell Cycling ◽  
Induced Apoptosis

Abstract In the multicentric ALL-BFM (Berlin-Frankfurt-Munster) study, all patients are uniformly treated during the first week of induction therapy which uses glucocorticoids (GC) as the principal therapeutic agent. The GC response assessed at day 8 of therapy provides one of the basic parameters for further risk stratification. In spite of the clinical significance, molecular mechanisms of GC action in vivo are largely unknown. Our recent genome-wide analysis of gene expression in blasts persisting during induction therapy identified a common set of genes differentially expressed in blasts at day 8 (d8) and at diagnosis (d0) (n=457, false discovery rate <0.05). Expression changes indicated therapy-induced inhibition of cell cycling, expression shift towards normal mature B cells and downregulation of the apoptosis regulator Bcl-2. In the current study, we validated the key differences between d8 and d0 blasts at protein and cellular levels. DNA distribution and percentage of cycling blasts was determined by flow cytometry in a series of matched d8 and d0 samples (13 pts) and demonstrated the decreased proliferative activity of d8 cells (4.3-fold, p=0.014). Protein expression, investigated by flow cytometry in a total of 84 pts, demonstrated statistically significant expression decrease of the progenitor cell antigenes CD10, CD34 and TdT and expression increase of the B-cell antigene CD20 and the inflammatory response molecules CD11b and IFNGR1 (p<0.05). We were also able to confirm the lower expression values of the Bcl-2 protein in d8 blasts (p<0.05, n=57). Investigation of GC-sensitive B-lineage leukemia cell lines demonstrated that BCL-2 downregulation by GC was a pre-requisite of GC-induced apoptosis. Moreover, we found a considerable cross-correlation between viability, cell cycling and Bcl-2 expression levels. Upregulation of the Bcl-2 expression via IL-7 receptor signaling prevented GC-induced apoptosis in these cell lines. Collectively, GC therapy interferes with differentiation and proliferation programs in leukemic blasts which are closely related to the Bcl-2 specific apoptotic pathway.

scholarly journals Influence of Individual Radiosensitivity on the Hormesis Phenomenon: Toward a Mechanistic Explanation Based on the Nucleoshuttling of ATM Protein

Dose-Response ◽  
2020 ◽  
Vol 18 (2) ◽  
pp. 155932582091378
Author(s):  
Clément Devic ◽  
Mélanie L. Ferlazzo ◽  
Elise Berthel ◽  
Nicolas Foray
Keyword(s):  
Low Dose ◽  
Molecular Model ◽  
Dose Range ◽  
Mechanistic Explanation ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Cellular Models ◽  
Individual Radiosensitivity ◽  
Biological Interpretation ◽  
Atm Protein

Hormesis is a low-dose phenomenon that has been reported to occur, to different extents, in animals, plants, and microorganisms. However, a review of the literature shows that only a few reports describe it in humans. Also, the diversity of experimental protocols and cellular models used makes deciphering the mechanisms of hormesis difficult. In humans, hormesis mostly appears in the 20 to 75 mGy dose range and in nontransformed, radioresistant cells. In a previous paper by Devic et al, a biological interpretation of the adaptive response (AR) phenomenon was proposed using our model that is based on the radiation-induced nucleoshuttling of the ATM protein (the RIANS model). Here, we showed that the 20 to 75 mGy dose range corresponds to a maximum amount of ATM monomers diffusing into the nucleus, while no DNA double-strand breaks is produced by radiation. These ATM monomers are suggested to help in recognizing and repairing spontaneous DNA breaks accumulated in cells and contribute to reductions in genomic instability and aging. The RIANS model also permitted the biological interpretation of hypersensitivity to low doses (HRS)—another low-dose phenomenon. Hence, for the first time to our knowledge, hormesis, AR, and HRS can be explained using the same unified molecular model.

JS-K, a GST-Activated Nitric Oxide Generator, Induces DNA-Double Strand Breaks and Inhibits Growth and Survival of Multiple Myeloma Cells In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3453-3453
Author(s):  
Tanyel Kiziltepe ◽  
Teru Hideshima ◽  
Noopur Raje ◽  
Kenji Ishitsuka ◽  
Enrique M. Ocio ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Multiple Myeloma ◽  
Double Strand Breaks ◽  
Cell Surface Expression ◽  
Dna Double Strand Breaks ◽  
Normal Peripheral Blood ◽  
Strand Breaks ◽  
Induced Apoptosis

Abstract JS-K (O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) is a diazeniumdiolate class of prodrug which is designed to release nitric oxide (NO·) on reaction with glutathione S-transferases (GST). GST has been shown to be overexpressed in a broad spectrum of tumor cells. Therefore, JS-K can possibly turn GST overexpression to the tumor’s disadvantage by generating high intracellular concentrations of cytotoxic NO·. Multiple myeloma (MM) is currently an incurable hematological malignancy where new treatment options are urgently needed. In this study we investigated the cytotoxicity of JS-K in MM in vitro and in vivo. JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient MM cells (IC50: 0.3–2.5 mM). Importantly, no significant cytotoxic effects of JS-K at these doses were observed in normal peripheral blood mononuclear cells. JS-K treatment induced apoptosis in MM cells which was associated with PARP, caspase 8, and caspase 9 cleavage; increased cell surface expression of Fas/CD95; Mcl-1 cleavage; Bcl-2 phosphorylation; as well as mitochondrial cyt c, AIF, and EndoG release. Moreover, JS-K could overcome the survival and growth advantages conferred by exogenous IL-6 and IGF-1, or by adherence of MM cells to bone marrow stromal cells. Flow cytometry experiments revealed significant NO· generation in JS-K-treated MM cells. Since NO· is known to cause DNA double strand breaks (DSB), we hypothesized that JS-K induces DSB in MM cells and confirmed DSB formation by neutral comet assay. We further showed that JS-K also activated DNA damage response pathways as evidenced by H2AX, Chk2 and p53 phosphorylation. In addition, JNK was also activated by JS-K treatment in MM cells, and inhibition of JNK significantly decreased JS-K-induced cytotoxicity, suggesting that JS-K induced apoptosis is mediated via JNK signaling. Finally, JS-K was also significantly effective in inhibiting tumor growth and prolonging median survival (p < 0.01) in a human plasmacytoma xenograft mouse model. Analysis of tumors harvested from treated animals showed that JS-K induced apoptosis and decreased angiogenesis in vivo. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.

Role of ET(A) and ET(B) receptors in endothelin-1-induced adrenal catecholamine secretion in vivo

1997 ◽  
Vol 272 (4) ◽  
pp. R1290-R1297 ◽  
Author(s):  
N. Yamaguchi
Keyword(s):  
High Performance ◽  
Low Dose ◽  
Dose Range ◽  
Control Group ◽  
High Dose ◽  
Dependent Manner ◽  
Experimental Conditions ◽  
Chromatography Method ◽  
Liquid Chromatography Method

The present study was designed to test whether endothelin (ET) A and/or B receptors in the adrenal medulla are functionally involved in ET-1-induced catecholamine (CA) release in anesthetized dogs. ET-1 was locally infused into the gland via the left adrenolumbar artery. Plasma CA in adrenal venous and aortic blood was determined by a high-performance liquid chromatography method. In the control group, the local infusion of ET-1 (0.5 microg, 0.4 microM) resulted in a significant increase in CA output. In the presence of a low dose of BQ-123 (5 microg, 16.4 microM), the ET-1-induced CA response was significantly attenuated by approximately 80%. With a high dose of BQ-123 (50 microg, 164 microM), the CA response was further blocked by approximately 95%. This inhibition was significantly greater than that obtained with the low dose of BQ-123. By contrast, a low dose of BQ-788 (5 microg, 15.1 microM) did not significantly affect the CA response. With a high dose of BQ-788 (50 microg, 151 microM), the CA response was only partially inhibited by approximately 70%. The results indicate that BQ-123 significantly inhibited ET-1-induced adrenal CA release in a dose-dependent manner. With the low doses, the CA response was markedly inhibited by BQ-123 but remained unchanged in the presence of BQ-788. Moreover, the high dose of BQ-123 virtually abolished the CA response, whereas BQ-788 failed to do so within the dose range tested. The present study suggests that the ET(A) receptor may play a predominant role in mediating the ET-1-induced CA secretion in the canine adrenal gland in vivo, although the possible involvement of the ET(B) receptor could not completely be excluded under the present experimental conditions.

Evodiamine Prevents Glioma Growth, Induces Glioblastoma Cell Apoptosis and Cell Cycle Arrest through JNK Activation

2017 ◽  
Vol 45 (04) ◽  
pp. 879-899 ◽  
Author(s):  
Wen-Shin Wu ◽  
Chih-Chiang Chien ◽  
Kao-Hui Liu ◽  
Yen-Chou Chen ◽  
Wen-Ta Chiu
Keyword(s):  
P53 Protein ◽  
Glioma Cells ◽  
Cyclin B1 ◽  
C6 Glioma Cells ◽  
Glioblastoma Cells ◽  
Anticancer Activities ◽  
Jnk Inhibitors ◽  
Induced Apoptosis

Evodiamine (EVO) is an active medicinal compound derived from the traditional herbal medicine Evodia rutaecarpa. It has been reported that evodiamine has several beneficial biological properties, including anticancer and anti-inflammatory activities. However, the in vitro and in vivo anticancer activities of EVO against the growth of glioblastoma cells remain undefined. EVO induced significant decreases in the viability of U87 and C6 glioma cells, but not of primary astrocytes, according with the occurrence of apoptotic characteristics including DNA ladders, caspase-3 and poly(ADP ribose) polymerase (PARP) protein cleavage, and hypodiploid cells. The disruption of the mitochondrial membrane potential (MMP) was detected, and it was found that the peptidyl caspase-9 inhibitor, Z-LEHD-FMK, significantly prevented glioma cells from EVO-induced apoptosis. Increased c-Jun N-terminal kinase (JNK) protein phosphorylation by EVO was observed, and the addition of JNK inhibitors, SP600125 and JNKI inhibited the EVO-induced apoptosis was inhibited. Additionally, EVO treatment induced G2/M arrest with increased polymerized tubulin protein expression in U87 and C6 cells. Elevated expressions of the cyclin B1, p53, and phosphorylated (p)-p53 proteins were detected in EVO-treated glioma cells, and these were inhibited by JNK inhibitors. An in vivo study showed that EVO significantly reduced the growth of gliomas elicited by the subcutaneous injection of U87 cells with increases in cyclin B1, p53, and p-p53 protein expressions in tumors. An analysis of eight EVO-related chemicals showed that alkyl groups at position 14 in EVO are important for its anti-glioma effects which involve both apoptosis and G2/M arrest. Evidence is provided that supports EVO induction of apoptosis and G2/M arrest via the activation of JNK-mediated gene expression and disruption of MMP in glioblastoma cells. EVO was shown to penetrate the blood–brain barrier; EVO is therefore predicted to be a promising compound for the chemotherapy of glioblastomas and deserves further investigations.

scholarly journals Requirements for Identification of Low Dose and Non-Linear Mutagenic Responses to Ionising Radiation

Dose-Response ◽  
2007 ◽  
Vol 5 (4) ◽  
pp. dose-response.0 ◽  
Author(s):  
Pamela J. Sykes ◽  
Tanya K. Day
Keyword(s):  
Low Dose ◽  
Threshold Model ◽  
Dose Range ◽  
Cellular Transformation ◽  
Low Dose Radiation ◽  
Spontaneous Frequency ◽  
High Doses ◽  
Dose Radiation

Cancer results from multiple changes in gene expression that can occur both genetically and epigenetically. High doses of radiation can lead to mutations and cancer. At high doses the number of mutations caused by radiation is essentially linear with dose. Low dose radiation induced protective responses observed for cancer in vivo and cellular transformation in vitro would predict that hormetic responses would also be observed in mutation assays. Although there are a large number of different mutation assays available, very few are able to detect changes in mutation frequency in response to very low doses of DNA damaging agents. The easiest way to cope with this lack of data in the low dose range is to invoke a linear-no-threshold model for risk assessment. The reasons for the lack of data are discussed. In order to identify hormetic mutation responses, assays need to have a spontaneous frequency that is high enough to enable a reduction below spontaneous frequency to be detected in a feasible number of scored cells and also need to be able to identify both genetic and epigenetic changes. The pKZ1 chromosomal inversion assay fits the criteria for detecting hormetic responses to low dose radiation.

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